This invention relates to a human stromelysin-1 (i.e., HSL-1) promoter and uses therefor. In particular, the invention relates to a purified DNA containing a functional HSL-1 promoter region that corresponds in structure to the native genomic form of this promoter, to an expression vector containing a portion of the purified promoter DNA operatively linked to a heterologous DNA sequence encoding a detectable gene product, and to the use of a host cell carrying the expression vector for drug screening and other pharmaceutical applications.
The stromelysin-1 promoter regulates the expression of the human stromelysin-1 gene, which encode major matrix metalloproteinase (MMP) of connective tissues. Stromelysin-2 and stromelysin-3 encoded by different genes and will not be discussed further here.
Stromelysin-1 degrades a broad spectrum of extracellular matrixother pharmaceutical applications. Components present in interstitial connective tissues, basement membranes, linings of the joints, and cartilage (e.g., proteoglycans, laminin, collagen IV, and fibronectin). It is currently reported that stromelysin indirectly stimulates the degradation of fibrillar collagens via proteolytic activation of procollagenases.
The controlled expression of stromelysin-1 is essential for tissue remodeling during normal development, wound healing, growth, aging and cellular responses to inflammation. Excessive matrix degradation contributes to the pathogenesis of a variety of disease-states, including rheumatoid- and osteoarthritis, bone resorption disease, abnormal angiogenesis, tumor invasion and metastasis, corneal ulceration and complications of diabetes. Thus an understanding of the regulation of stromelysin-1 promoter function has important consequences for the therapy of human diseases which involve increased expression of MMPs.
Aspects of stromelysin-1 promoter function have been studied using a portion of a 1.3 kb sequence isolated by Quinones et al from a circa 10 kb sequence upstream of the 5xe2x80x2 transcription start site in a genomic clone of stromelysin-1 (Quinones et al, J. Biol. Chem. 264: 8339-8344 (1989); Buttice et al, Nucl. Acids Res. 19: 3723-3731 (1991); Diaz-Meco et al (J.Biol.Chem. 266: 22597-22602 (1991)), and a 0.30 kb sequence of a promoter region in a genomic clone isolated by Sirum and Brinkerhoff (Sirum and Brinckerhoff, Biochemistry 28: 8691-8698 (1989)).
The aforementioned 1.3 kb sequence extends 5xe2x80x2 from the first ATG codon through the transcription start site to an Sst I site 1.3 kb upstream (xe2x88x921303 to +66). A portion of this sequence (xe2x88x921303 to xe2x88x9211) was cloned into a human growth hormone reporter gene vector, sequenced and studied. In transient transfection assays, it was determined that the 1.3 kb fragment contains DNA elements required for IL-1xcex2 induction and dexamethasone suppression, and that the level of induced expression varies inversely with fragment length. (Quinones et al, 1989). Responsiveness to PDGF and PC-PLC induction was localized to the region between xe2x88x921279 and xe2x88x921240. Diaz-Meco et al, J.Biol.Chem. 266: 22597-22602 (1991). Buttice et al cloned the portion xe2x88x921303 to +4 into a human growth hormone reporter gene vector (henceforth referred to as xe2x88x921303+4HGH) and demonstrated by transient transfection assays in mouse F9, HFF, HeLa and HepG2 cells that the AP-1 site (xe2x88x9270 to xe2x88x9264) was required for basalxe2x80x94but not for TPA-induced reporter gene expression. Buttice et al, Nucl. Acids Res. 19: 3723-3731 (1991).
Sirum and Brinckerhoff isolated human stromelysin-1 genomic clones and showed that the cloned DNA and human lymphocyte genomic DNA yielded the same-sized restriction fragments when digested with the restriction enzyme EcoRI (i.e. fragments of 1.4, 2.6 and 6 kb were detected by hybridization to a stromelysin cDNA probe). An EcoRI-DdeI promoter fragment from xe2x88x92325 to xe2x88x9218 relative to the AGT translation start codon was cloned into a CAT reporter gene vector. The vector was subsequently used to demonstrate that elements required for PMA, EGF and IL-1xcex2 induction were contained in about 270 bp of 5xe2x80x2 flanking DNA.
Reporter gene constructs incorporating the above promoter fragments have provided confirmation that basal expression of stromelysin-1 is low in normal tissues, but can increase markedly in the presence of known inducers. H.Birkedal-Hansen et al, Critical Reviews in Oral Biology and Medicine, 4: 197-250 (1993); Buttice, G. and Kurkinen, M., J. Biol. Chem. 268: 7196-7204 (1992); Diaz-Meco, M. T. et al, J. Biol. Chem. 266: 22597-22602 (1991); Sirum-Connolly, K. and Brinckerhoff, C. E., Nucleic Acids Res. 19: 335-341 (1991). As well, the induction of stromelysin-1 gene expression can be inhibited by agents such as transforming growth factor-xcex2, dexamethasone, retinoic acid, and cAMP. Birkedal-Hansen et al, (1993); Frisch, S. M. and Ruley, H. E., J. Biol. Chem. 262: 16300-16304 (1987).
Regulatory sequences that have been identified thus far appear to be highly conserved between species (see Matrisian et al, Mol. Cell Biol. 6: 1679-1686 (1986)(rat transin/stromelysin-1); Frisch and Ruley, J. Biol. Chem. 262: 16300-16304 (1987)), particularly in the region between xe2x88x92480 and transcription start site. However, as the present invention discloses, the region upstream of xe2x88x92480 contains a novel 1 kb sequence which is present in the genomic promoter in its native state but is absent from the 1.3 kb promoter region disclosed in Quinones et al (1989). PCR analysis and EcoRI restriction mapping both confirm the presence of this novel sequence. Also disclosed is the reappearance of the sequence of the published clone upstream of the 1 kb sequence, but in an inverse orientation.
Thus in addition to the functional regulatory sequences defined previously, the present invention possesses a native promoter architecture. The three dimensional relationship between enhancers and promoters is likely to be important for tissue- and developmental stage-specific transcription of MMP genes. For example, it is known that stromelysin-1 is coordinately expressed with other MMP genes in a cell type-specific manner. Birkedal-Hansen et al, (1993); Buttice et al (1991).
Given that the stromelysin-1 promoter is relatively quiescent in normal adult tissues but is activated under conditions of excessive extracellular matrix degradation (e.g., in inflammatory conditions, in some metastasizing tumors, and in rheumatoid arthritis and other diseases characterized by increased expression of metalloproteinases), the present invention is useful in ameliorating these conditions using gene therapy techniques, and in screening for pharmacologic agents that act transcriptionally to modulate stromelysin-1 levels.
An aspect of the present invention is to provide an isolated human stromelysin-1 promoter having a native genomic DNA promoter sequence of at least 1.7 kb upstream of the transcription start site.
Another aspect of the present invention is to provide a recombinant DNA molecule identified by SEQ ID NO:1, its complementary strand, high stringency-hybridizable nucleotide sequences thereof, and functional segments of the foregoing DNA sequences at least 1.47 kb in length.
Yet another aspect of the present invention is to provide the recombinant DNA sequence identified by SEQ ID NO:1, or a functional portion thereof, in a transfer vector for use in transferring the sequence to a host cell.
Another aspect of the present invention is to provide recombinant expression systems in which the DNA sequence (SEQ ID NO:1), or a functional portion thereof, is operably linked to a heterologous gene encoding a detectable product, preferably a reporter gene such as luciferase or xcex2-galactosidase, most preferably the tumor necrosis factor-xcex1 receptor, TNFxcex1R, or the tissue inhibitor of matrix metalloproteinases, TIMP. Still another object of the present invention is to provide such an expression system in a eukaryotic host cell that can be induced by inflammatory mediators (e.g., TNFxcex1, IL-1xcex2) and phorbol-type tumor promoters to express the product of the heterologous gene contained inserted into the vector. Yet another object of the invention is to provide a method of modulating extracellular levels of TNF-xcex1 and MMP activity by using the recombinant expression system to produce biologically active TNF-xcex1R and TIMP.
Another aspect of the invention is to provide a recombinant expression system comprising at least 1.47 kb of the cloned promoter of the present invention operably linked to a reporter gene and contained within an appropriate host cell that can be used to assay for stromelysin-1 promoter activity, and a method for using this assay to screen for pharmacologic activators and inhibitors of stromelysin-1 promoter activity.
It should be understood that the foregoing general description and the following detailed description are intended to be exemplary and explanatory only, and not restrictive of the invention, as claimed.